Tumor Differentiation is the Dominant Prognostic Factor for Patients with Colorectal Neuroendocrine Neoplasms with Distant Metastasis

Purpose Colorectal neuroendocrine neoplasms (NENs) are rare tumors. The prognosis and prognostic factors of metastatic colorectal NENs have not been fully elucidated. Methods We retrospectively enrolled 77 consecutive patients diagnosed with colorectal NENs with synchronous distant metastases between 2000 and 2021. All patients were assigned to the neuroendocrine tumor (NET) group or the neuroendocrine carcinoma (NEC) group based on histological differentiation. Propensity score matching (PSM) was performed to minimize confounding bias. The Kaplan–Meier method was used to calculate the survival rates. Univariate and multivariate logistic regression analyses were performed to identify prognostic factors. Results In total, 35 (45.5%) and 42 (54.5%) patients had well-differentiated NETs and poorly differentiated NECs, respectively. The median overall survival (OS) was 26 months for the entire cohort, and the 1-year, 3-year, and 5-year OS rates were 69.4%, 41.4%, and 27.8%, respectively. In the subgroup analysis, the median OS was 62 and 10 months for NETs and NECs, respectively. Univariate analysis demonstrated that patients with a primary tumor located in the colon, ulcerative tumors and poorly differentiated tumors were at higher risk for poorer progression-free survival (PFS) and OS. However, only histological differentiation was identified as an independent factor affecting OS (hazard ratio (HR) = 8.28, 95% confidence interval (CI): 2.98–23.01, P < 0.001) in multivariate analysis. After PSM, histological differentiation was further confirmed as the dominant factor affecting OS (HR = 6.09, 95% CI: 1.96–18.95, P=0.002)). Conclusion Histological differentiation was the most dominant prognostic factor in patients with metastatic colorectal NENs. Patients with well-differentiated NETs had a good chance of long-term survival.


Introduction
Colorectal neuroendocrine neoplasms (NENs) are a rare subtype of colorectal tumors. However, a rapidly increasing incidence of colorectal NENs has been observed in recent decades, owing to the utilization of colonoscopy screening [1][2][3]. Colorectal NENs are a group of highly heterogeneous tumors with signifcantly diferent clinical features and outcomes based on their pathological manifestations [4]. According to the 2019 edition of the WHO classifcation system, colorectal NENs are categorized into G1, G2, and G3 NENs based on the Ki-67 index and mitotic count. All G1 and G2 NENs are regarded as well-diferentiated and are named G1 and G2 neuroendocrine tumors (NETs), respectively. However, for G3 NENs, well-diferentiated G3 NENs are termed G3 NETs, whereas poorly diferentiated G3 NENs are termed neuroendocrine carcinomas (NECs) [5,6].
However, the rates of distant metastasis vary widely from G1 to G3 NENs. For patients with G1 and G2 NENs, metastatic NENs are found in only 0.3% and 6.3% of these patients, respectively [12]. More than half of patients with G3 NENs show distant metastasis at the date of diagnosis [13,14]. Metastatic G3 colorectal NENs are highly aggressive and malignant, with a median overall survival time of only 4-10 months [14,15].
Owing to the rarity of metastatic colorectal NENs, there is still no widely acknowledged consensus regarding optimal treatment strategies. In addition, the prognostic factors for these patients have not been well understood in previous studies. In the present study, we aimed to evaluate the clinical outcomes of metastatic colorectal NENs and identify important prognostic factors that will aid in the management of these tumors.

Study Design and Participants.
Tis study received approval from the Ethics Committee of the National Cancer Center and was performed in accordance with the Declaration of Helsinki of the World Medical Association. We conducted a retrospective study and enrolled 69 and 8 patients diagnosed with metastatic colorectal NENs between 2000 and 2021 from the National Cancer Center/Cancer Hospital, the Chinese Academy of Medical Sciences and the Strategic Support Force Medical Center, respectively. Te inclusion criteria were as follows: (1) all cases were pathologically confrmed as colorectal NENs, and (2) all cases presented with synchronous metastatic disease at the initial diagnosis. Te exclusion criteria were as follows: (1) patients sufering from another malignant tumor and (2) patients with incomplete clinicopathological data. Finally, a total of 77 eligible patients were included in the present study. We extracted clinicopathological information from the electronic medical database and obtained survival data through telephone calls or outpatient visits. Te last date of follow-up was April 30, 2022.
Te primary outcomes were the survival outcomes of these advanced patients, including progression-free survival (PFS) and overall survival (OS). Te PFS was calculated between the initial anticancer treatment date and the tumor progression date based on imaging techniques. OS was calculated between the date of initial anticancer treatment and the date of cancer-specifc death. Te secondary outcomes were the prognostic factors that afected PFS and OS.

Statistical
Analysis. Continuous data that followed a normal distribution were expressed as means and standard deviations (SDs), and then they were compared using a t test. Continuous data that did not follow a normal distribution are presented as medians and ranges, and they were compared using the Mann-Whitney U test. Qualitative and ordinal data are presented as counts and percentages, and they were compared using the X 2 test or Fisher's exact test for categorical data and the Mann-Whitney U test for ordinal data. To reduce the imbalance between the NET group and the NEC group, propensity score matching (PSM) was performed by ftting a logistic regression model and setting the caliper at 0.05. One-to-one pair matching was performed without replacement, and 20 matched pairs were selected ( Figure 1). Te covariates included the primary tumor location, size, shape, TNM T stage, TNM N stage, presence of extrahepatic metastasis, and surgical treatment. PFS and OS were calculated using Kaplan-Meier curves and compared using the log-rank test. Univariate and multivariate logistic regression analyses were conducted to identify independent prognostic factors. All data calculations and analyses were performed using Statistical Package for the Social Sciences (SPSS version 24.0; IBM Corp., Armonk, NY). Statistical signifcance was set at a two-sidedP value < 0.05.

Clinicopathological Characteristics.
Te clinical and pathological data are shown in Table 1. From 2000 to 2021, a total of 77 consecutive patients were retrospectively included in this study with an average age of 57.7 ± 12.2 years. In total, 43 (55.8%) and 34 (44.2%) patients were males and females, respectively. Hematochezia (35.1%) and changes in bowel habits (35.1%) were the most common clinical manifestations, followed by abdominal pain (27.3%) and distention (10.4%). No cases of carcinoid syndrome were found. Remarkably, 9 (11.7%) patients were asymptomatic and diagnosed through routine physical examination. With regard to the location of the primary tumor, more than half (72.7%) of the patients had their primary tumor located in the rectum, followed by the ascending colon (10.4%) and sigmoid colon (7.8%). Te liver was the most common organ of distant metastasis (80.5%), and 20 (26.0%) and 17 (22.1%) individuals presented with distant lymph node and bone metastases, respectively. Surprisingly, lung metastasis was less likely than expected, and only 1 (1.3%) patient sufered from lung metastasis.
Te treatment-modality data for these patients are shown in Table 1. A total of 35 (45.5%) individuals received surgical resection, including 12 (15.6%) individuals who underwent radical excision and 23 (29.9%) individuals who underwent palliative excision. Most (83.1%) of the cohort received systematic chemotherapy, and only 4 (5.2%) patients received radiotherapy. Four (5.2%) patients refused to receive any treatment.

Propensity Score Matching.
Te diferences in the clinicopathological variables between patients with metastatic colorectal NETs or NECs before and after PSM are detailed in Table 2. In the original cohort, signifcant differences regarding primary tumor location, size, shape, invasion depth, and presence of extrahepatic metastasis were observed between NETs and NECs. After PSM, these factors were comparable between the NET group and the NEC group.

Oncological Outcomes.
A median follow-up duration of 15 (range 1-88) months was obtained in this study. Six patients were lost to follow-up due to a loss of communication, leading to a follow-up rate of 92.2%. Te median PFS was only 5 months, and the 1-year, 3-year, and 5-year PFS rates were 25.5%, 16.2%, and 16.2%, respectively ( Figure 2(a)). Te median OS was 26 months, and the 1-year, 3-year, and 5-year OS rates were 69.4%, 41.4%, and 27.8%, respectively ( Figure 2(b)).
Te univariate and multivariate analyses of the risk factors afecting PFS and OS in the original cohort are presented in Table 3. We demonstrated that the histological diferentiation (P � 0.001), location (P � 0.010), and macroscopic morphology (P � 0.007) of the primary tumor were associated with PFS using univariate Cox proportional hazards regression analysis. However, none of these variables were confrmed as independent risk factors afecting PFS in subsequent multivariate analysis. Using univariate Cox proportional hazards regression analysis, we found that histological diferentiation (P < 0.001), location (P � 0.003), and macroscopic morphology (P < 0.001) of the primary tumor were associated with OS. Subsequent multivariate analysis indicated that tumor diferentiation was the only prognostic factor that signifcantly afected OS ((hazard ratio (HR) � 8.28, 95% confdence interval (CI): 2.98-23.01, P < 0.001)).
For patients with colorectal NETs, the median PFS was 10 months, and the 1-year and 3-year PFS rates were 41.7% and 32.5%, respectively. However, for patients with colorectal NECs, the median PFS was only 2 months, and the 1year and 3-year PFS rates were 11.2% and 7.5%, respectively (Figure 3(a)). Regarding OS, patients with colorectal NETs reached a satisfactory median OS of 62 months, and the 1year and 3-year OS rates were 97.1% and 79.3%, respectively. In contrast, the patients with colorectal NECs had signifcantly decreased OS; the median OS was only 10 months, and the 1-year and 3-year OS rates were 46.6% and 30.8%, respectively (Figure 3(b)).
Te univariate and multivariate analyses of the risk factors afecting PFS and OS in the matched cohort are presented in Table 4. No signifcant variables afecting PFS were found. Tumor diferentiation was further confrmed as the only prognostic factor that signifcantly afected OS (HR � 6.09, 95% CI: 1.96-18.95, P � 0.002)). Te survival curves of PFS and OS in the matched cohorts are shown in Figure 3. Tere was no signifcant diference in PFS between NETs and NECs. However, the median OS was 88 months in the NET group and 13 months in the NEC group, and this diference was statistically signifcant (P < 0.001).

Discussion
Colorectal NENs are a group of rare tumors with high heterogeneity, and the vast majority of them are indolent, well-diferentiated, and localized lesions at an early stage, which can be cured through endoscopic resection [16,17]. Metastatic NENs represent only a small fraction of all colorectal NENs. In a study on 5457 patients with colorectal NENs from the National Cancer Database (NCDB), Chagpar et al. reported that only 299 (5.5%) patients demonstrated distant metastasis [8]. In another study on 607 patients with hindgut NENs, Kim et al. reported that only 42 (6.9%) patients presented extensive disease [9]. Although distant metastasis only occurs in a small portion of all colorectal NENs, the rates vary widely based on their grade and differentiation. In a study that included 9926 NENs of the colon and rectum from the Surveillance, Epidemiology, and End Results (SEER) database, Ding et al. found that only 1092 (11.0%) individuals sufered from metastatic disease; however, in a subgroup analysis, metastatic disease developed in 3.6% and 41.1% of patients with well-diferentiated NETs and poorly diferentiated NECs, respectively [10]. Another report from the C-NET study, a Japanese multicenter prospective study of colorectal NETs, included 500 colorectal NETs, and only 3 (0.6%) of them presented metastatic disease [18]. However, most previous studies have shown that more than half of colorectal NECs have distant metastases at the date of initial diagnosis. Owing to the rarity of metastatic colorectal NETs, few studies have described the prognosis of patients and compared the diference between NETs and NECs.
Te underlying genetic and molecular mechanisms as well as the risk factors for distant metastasis have not been fully elucidated in previous studies, especially for colorectal NETs. Generally, NETs with a size of <1 cm are at an extremely low risk of metastasis and can be endoscopically resected with a favorable prognosis [19,20]. However, in the present study, we observed 6 metastatic NETs with primary lesions smaller than 1 cm. Few previous studies have focused on metastatic NETs originating from diminutive primary tumors. One available study has reported that 1% of all colorectal NENs smaller than 1 cm are at risk of distant metastasis [21]. Another study has reported that 3.7% of all colorectal NENs smaller than 2 cm may develop metastatic disease [22]. It remains unknown why these diminutive and indolent NETs develop distant metastases, thus requiring further exploration. However, these results may indicate that     International Journal of Endocrinology even for small and localized NETs, it is necessary to evaluate patients with whole-body imaging to exclude metastatic disease before endoscopic therapy, which may be missed when following current guidelines in clinical practice [17,23]. Te present cohort had a median OS of 26 months with 3-year and 5-year OS rates of 41.4% and 27.8%, respectively. In a real-world data retrospective cohort study from the SEER database with 9732 patients with metastatic colorectal tumors, Han et al. reported that 80.95% of these patients had colorectal adenocarcinomas; they reported a median OS of 23 months, and the 3-year and 5-year OS rates were 32.4% and 18.4%, respectively [24]. Compared to the overall population of people with colorectal cancer, colorectal NENs may have a higher probability of long-term survival. However, colorectal NENs are a group of highly   [14,15]. Te signifcant differences in survival outcomes indicated that accurate determination of tumor diferentiation is crucial in predicting prognosis. However, assessing the degree of tumor diferentiation is challenging. In earlier studies, all G1 and G2 NENs were regarded as well-diferentiated NENs, while all G3 NENs were acknowledged as poorly diferentiated NECs, which was based on the World Health Organization (WHO) 2010 classifcation and nomenclature system for digestive NENs [17]. Recent studies have described a new entity of high-grade, well-diferentiated NENs (NET G3) and separated them from the prior G3 NENs [25]. In the recent 2019 edition of the WHO classifcation system, G1, G2, and G3 well-diferentiated NENs are named NETs, while G3 poorly diferentiated NENs are termed NECs [5,6]. However, it is challenging to distinguish G3 NETs from NECs in many cases based only on morphology diferentiation. Genetic information and proliferative activity are often taken into account. NENs with mutations in KRAS, BRAF, p53, and Rb1 or with a Ki-67 index greater than 70%-80% are usually diagnosed as NECs [26,27].

International Journal of Endocrinology
Tere are limited reports regarding the genetic discrepancies between colorectal NETs and NECs. Chen et al. extracted data from the American Association for Cancer Research (AACR) Project Genomics, Evidence, Neoplasia, Information, and Exchange (GENIE) database and accessed genetic data from 83 colorectal NECs, 276 gastrointestinal NETs, and 6476 colorectal adenocarcinomas. Tese authors demonstrated that colorectal NECs presented a signifcantly higher tumor mutation burden (TMB) (5.16 versus 1.43) and a higher somatic mutation rate of TP53 (65.5% versus 6.8%) than colorectal NETs. With regard to copy number alteration (CNA), colorectal NECs usually had CNA frequently in chromosomes 8, 9, 13, 16, 17, and 20 with a gain of MYC (12.3%), loss of RB1 (10.7%), and loss of PTEN (5.4%) being the most common CNAs, while colorectal NETs often had the most CNAs occurring in chromosomes 3,9,11,12,17, and 18 with a low CNA frequency [28]. Chen et al. concluded that colorectal NECs are more similar to colorectal adenocarcinomas. Te genetic diference between colorectal NETs and NECs may explain their signifcantly diferent clinical outcomes and further guide individualized treatment for these advanced patients.
Te present study had aforementioned limitations. Due to the retrospective nature of the present study, bias arising from the collection of information could not be completely avoided. Further prospective studies are needed to delineate the clinicopathological and genetic characteristics of NENs as well as the clinical outcomes of patients. Te second limitation was the small sample size of the present study.
Owing to the rarity of colorectal NENs and the low proportion of metastatic disease, we enrolled a limited number of patients over a period of 20 years. Multicenter studies from more hospitals should be performed to include more patients in further studies.

Conclusion
Metastatic colorectal NENs are a group of highly heterogeneous tumors, and histological diferentiation is the most important prognostic factor. Metastatic patients with welldiferentiated colorectal NETs have a favorable prognosis, while those with poorly diferentiated colorectal NECs have poor survival outcomes.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.